This invention generally relates to the field of new dyes, dye compositions, and methods of enumeration of reticulocytes by flow cytometry by employing fluorescent, RNA binding dyes.
Enumeration of reticulocytes, i.e., the immature erythrocytes, in human peripheral blood is a valuable component of diagnostic hematology. Such enumeration is useful in the diagnoses of hemorrhage, anemia, monitoring bone marrow transplantation and for monitoring patients undergoing chemotherapy and other disorders involving blood cell production [U.S. Pat. No. 5,360,739; H. Shapiro, Practical Flow Cytometry, 3rd edit., 1995; Wiley-Liss, New York; Davis et al, (1990) Pathobiol., 58:99-106; Hoy, (1990) Bailliere""s Clin. Haemat., 3:977-988; H. J. Tanke, Reticulocytes and Mature Erythrocytes in Flow Cytometry in Haematology (1992) Academic Press Ltd, pp. 75-93]. Because reticulocytes contain ribonucleic acid (RNA), if stained with RNA binding excitable dyes, these cells fluoresce when illuminated by a light source of appropriate wavelength. RNA binding dyes have been used to distinguish reticulocytes from mature red blood cells (RBCs) which lack RNA.
Distribution of fluorescence intensities of a relatively large reticulocyte population can be determined by flow cytometry in a fast and reliable manner, and different maturation stages of reticulocytes, as reflected by differences in RNA content, can be distinguished.
The use of red-excitable dyes is desirable because such dyes are detected by excitation with relatively inexpensive diode or HeNe lasers. However, initial efforts in the prior art to employ diode lasers and red-excited dyes for rapid flow cytometric analyses of reticulocytes were not successful. Yamamoto, U.S. Pat. No. 5,563,070 suggested that the addition of large quantities of TO-PRO-3, a red-excitable dye, followed by a 30 minute incubation, stained RNA inside living reticulocytes. Such a method, however, is not practical for routine analysis of reticulocytes in clinical laboratories that require high sample throughput because sample preparation time is long and cost per test is high due to the large amount of dye required to stain each sample.
A red-excitable dye called Thiazole Blue (TB) has been described in U.S. Pat. No. 4,957,870. However, as described in this patent (U.S. Pat. No. 4,957,870), this dye also requires long periods of incubation, of about 30 minutes.
Akai et al., U.S. Pat. No. 5,821,127 have also described the preparation of fluorescent dyes which are capable of detecting reticulocytes using inexpensive detectors via fluorescence in the red region. However the samples require incubation at elevated temperatures of about 40xc2x0 C.
More recently, U.S. Pat. No. 5,994,138, described staining reticulocytes via the use of a red-excitable dye in combination with a detergent and an ionophore at elevated temperatures of about 35xc2x0 C. However, staining reticulocytes was not successful when ambient temperatures were utilized.
Fan et. al (U.S. Pat. Nos. 5,411,891, 5,360,739) describes clearly that the specific binding constant between a dye and reticulocyte RNA and the rate of penetration of the dye are different for each dye and that it is impossible to predict under what conditions a particular dye may rapidly penetrate red cell membrane and stain reticulocytes. This was further supported by Akai et. al (U.S. Pat. No. 5,821,127).
Thus, there exists a need in the art for dyes, compositions and methods which enable rapid staining of intracellular RNA at room temperatures via the use of dyes that are excitable in the red region and can use inexpensive and readily available red-illumination instruments. In addition there exists a need in the art for dye compositions and methods that are applicable not only to red-excitable dyes, but also to dyes excitable at other wavelengths, such as in the blue wavelength. By doing so, ready and accurate detection of reticulocytes can be accomplished without particular restriction of the excitation wavelength.
In one aspect, the present invention provides novel dyes of the formula: 
wherein n is 0, 1, 2, or 3; R1 is H, alkyl, or an alkoxy group; R2 is CH2(CH2)mOH,
wherein m is 0 to 3; X is O, S, or C(CH3)2; R is CH3, CH(CH3)2, CH2CH2OH, alkyl, alkysulfonate, or hydroxyalkyl; and Bxe2x88x92 is a counteranion.
In another aspect, the invention provides a reagent containing a dye of the invention and a solvent.
In yet another aspect, the invention provides compositions and methods for facilitating rapid transport of dye molecules through a cell membrane. This composition contains the dye of this invention and at least one member selected from the group comprising a detergent, a surfactant, a sulfonic acid or a salt thereof.
Other aspects and advantages of this invention will be readily apparent from the detailed description of the invention.